Preparation and use of thiophosphonates and thio-analogues of phosphonoformic acid

ABSTRACT

Methods for converting phosphonates into thiophosphonates and specific thiophosphonate compounds so produced are disclosed and claimed. The methods start with a reaction mixture formed of a phosphonate compound, including one or more strong electron-withdrawing groups located adjacent to the phosphorus in the compound, a slight excess of Lawesson&#39;s reagent, and a polar aprotic solvent. The reaction mixture is heated until reaction is complete and may be followed with separation or hydrolyzation steps to produce thiophosphonic acids and their addition salts. One of these thio-analogues, thiophosphonoformic acid (TPFA) is particularly effective at inhibiting HIV replication and in treating mammals infected with HIV.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention is directed to new and useful processes for thelarge scale production of thio.analogues of phosphonoformic acid (PFA)and to the conversion of phosphonates into thiophosphonates in generalas well as to the thio-PFA (TPFA) compounds produced by theseprocedures. An additional aspect of the present invention relates to theuse of these thio-PFA compounds as antiviral agents which areparticularly effective against HIV.

2. Description of the Prior Art

Organic compounds of the general structure ##STR1## wherein X is oxygen(O) or sulphur (S) are known, respectively, as phosphonates andthiophosphonates. These compounds are implicated in a variety ofbiological processes and show promise in basic research for medical andagricultural uses including pesticides and antiviral compounds.Unfortunately, research involvingthiophosphonates is often hindered bythe extreme difficulty in producing even small quantities of thesephosphonate analogues. Moreover, economic methods for the large scaleproduction of thiophosphonates are virtually unknown.

For example, of particular interest to the present invention are thethio-analogues of phosphonoformic acid. Phosphonoformic acid (PFA) andits thio-analogue. thiophosphonoformic acid (TPFA) have the followinggeneral formulae and structures: ##STR2## Early efforts reportedlyproducing TPFA utilized the Michaelis-Becker reaction between thesodio-derivative of diethyl thiophosphite and ethyl chloroformate orchloroacetate, followed by the removal of the P-OEt groups withiodotrimethylsilane (ITMS) at high temperature over 48 hours. However,recent research has indicated that this method is not reproducible and,because of difficulties including the removal of the ethyl groups,produces mixtures of a variety of compounds rather than the desiredTPFA. Other proposed methods for the synthesis of TPFA are equallydifficult and expensive utilizing numerous steps with exceptionallylowproduct yields. Similar difficulties and expense are associated withthe production of the thio-analogues of other phosphonates as well.

Difficulties in producing usable quantities of thiophosphonates are notrestricted to commercial applications requiring large quantities ofproduct. Basic research involving these compounds also requires readilyavailable, pure materials. For example, as proposed and claimed by thepresent invention, TPFA shows great promise as an antiviral agent foruse in combatting HIV infection and AIDS in mammals. These propertiescould not be determined in the past due to the inability of the priorart methods to produce usable quantities of essentially pure TPFA.However, before discussing these antiviral properties in detail, ageneral understanding of antiviral therapy will be of assistance.

Unlike infectiousbacteria, which are functionally and physicallydistinct and can reproduce outside the cells of their host organisms,the simplicity of viruses makes them able to replicate only byphysically invading a host cell and co.opting its biochemical mechanismsto make new viral components. As a result of this intimate connectionwith the replication cycle of the host cell, viruses present few uniquebiochemical features which can be selectively attacked without poisoningthe host cell. As recently as the 1960's, it was believed that the onlystrategy for controlling viral infections was the development ofvaccines against specific viruses to forestall infection by stimulatingthe immune system of uninfected individuals in advance.

In spite of these problems, recent developments in the understanding ofthe details of viral functions have brought to light unique aspects ofviral activities which may provide targets for attack. This accumulatingbody of knowledge has made it possible to identify compounds that mayselectively interfere with these viral activities without poisoning thehost cells of the infected organism. For example, in both lytic viralinfections (those that spread rapidly throughout the population ofvulnerable cells, destroying them early in the illness) and persistentviral infections (those that do not always kill an infected cell) theviral agents complete their replication cycles through a number ofunique steps that an antiviral drug may interrupt.

Unfortunately, the present state of the art is such that antiviral drugsare only capable of attacking such viruses when they are replicating.Attacking a latent virus such as HIV which does not reproduce itselffollowing infection until reactivated by presently unknown factorswouldrequire distinguishing the viral genetic material from thesurrounding host genetic material and selectively destroying it. Thus.the current generation of antiviral drugs is only effective againstreplicating viruses.

Nonetheless, there are notable successes in the field of antiviral drugtherapy. An exemplary antiviral compound is acyclovir, a nucleosideanalogue which mimics the structure of a precursor of DNA Acyclovir hasbeen found to interfere with the viral enzymes thymidine kinase and DNApolymerase specific to some herpes viruses, thereby inhibiting thesynthesis of the viral DNA and ultimately viral replication itself.Similar antiviral effectiveness has been produced with a differentnucleoside analogue. ribavirin which interferes with a viral enzymecrucial to the synthesis of DNA and RNA as well as selectivelyinhibiting viral mRNA and thus the production of viral proteins. Thoughfar more effective against viral functions, ribavirin, like manyantiviral compounds, may also affect human cells and thus may be toxicto rapidly metabolizing cells such as blood cells, limiting itsapplicability and usefulness.

In spite of these and other antiviral success stories, the mostimportant current challenge for the development of antiviral compoundsis the need for an effective treatment against HIV, the viral cause ofthe AIDS pandemic. In contrast to the bleak epidemiological picture ofAIDS wherein potentially millions of people are believed to be infected,the accumulation of knowledge aboutHIV and its functions has beenunprecedentedly rapid. Though only identified in 1983, HIV is known tobe a retrovirus whose main target is the T4 lymphocyte, a white bloodcell which marshals the immune defenses of the infected host.Additionally, the virus also infects cells in the central nervoussystem.

After binding to a host cell, HIV penetrates the cell and exposes itsviral genetic material: a single strand of RNA. Accompanying the viralRNA is a viral enzyme known as reverse transcriptase which converts theviral genetic material into DNA which becomes integrated into thechromosomes of the infected host cell. The integrated viral genome or"provirus" remains latent until the host cell is stimulated and thendirects the synthesis of viral proteins andRNA which assemble to formnew HIV particles which burst from and destroy the host cell.

The current target for antiviral drug therapy against HIV replication isthe reverse transcription step which is crucial to the viral replicationyet irrelevant to the infected host cells. A variety of antiviral drugshave been shown to reduce the activity of HIV reverse transcription invitro to varying degrees. These compounds include azidothymidine (AZT),suramin, antimoniotungstate, dideoxynucleotides, and phosphonoformateAZT, has shown significant positive effects in large.scale clinicaltrials though major concerns remain about its considerable toxicity tobone.marrow cells.

Several researchers have indicated that the pyrophosphate analogues.phosphonoacetic acid (PAA) and phosphonoformic acid (PFA) possessantiviral properties in that they inhibit the replication of severalviruses including influenza virus A and herpes virus HSV.I. Research hasshown that these compounds have an inhibitory activity on the reversetranscriptase of influenza virus A and the DNA polymerase of HSV.I aswell as on the DNA polymerase of mammalian cells. (D. W. Hutchinson, G.Semple. and D. M. Thornton. Synthesis and Biochemical Properties of SomePyrophosphate Analogues, Biophosphates and Their Analogues Synthesis,Structure, Metabolism and Activity, K. S. Bruzik and W. J. Stec (Eds.).Elsevier Science Publishers, B. V., 1987, 441.450.)

Additionally, it has also been suggested in the art that thethio-analogues of phosphonoacetic acid (PAA) and phosphonoformic acid(PFA) may have potential as antiviral agents. (D. W. Hutchinson and S.Masson. The antiviral potential of compounds containing thethiophosphoryl group. I.R.C S. Medical Science 14 (1986) 176.177.)However, recent research by the inventor has raised significantquestions as to the veracity of such reports. It is believed that thereported activities of the alleged thio-PFA compounds discussed in theseprior art references are deceivingly incorrect as the prior art methodsfor preparing these compounds do not produce TPFA but, instead, producemixtures of different, unidentified compounds.

Moreover, as those skilled in the art will appreciate, further questionsas to the accuracy and basis of such unsupported speculation withrespect to the proposed properties of TPFA results from the fact thatthe inhibition of viral enzymes by such compounds in general is uniquelyspecific to the viral enzymes involved. Thus, it is impossible topredict the antiviral activity of a particular compound as that compoundmay or may not be effective against a particular virus. For example.acyclovir has proven to be beneficial in infection by Herpes virus, yetacyclovir.resistent strains of Herpes virus have been located.Similarly, Epstein-Barr virus (EBV) is relatively insensitive toacyclovir. Thus, it is clear that early signs of some antiviral activityare not indicative of a compound's effectiveness as an antiviral drug.

Further complicating matters, a compound which may inhibit viralactivity may also inhibit critical functions of the host cell and thusprove to be toxic to the host. As a result, antiviral compounds whichmay be effective in vitro may not be effective as antiviral agents invivo due to a lack of significant differences in their relativeinhibitory activities with respect to viral and host cell mechanisms.

Accordingly. it is a principal object of the present invention todisclose methods for the effective production of large quantities ofthio-analogues of PFA in order to facilitate the research andutilization of such compounds.

It is an additional object of the present invention to discloseProcesses for inexpensively producing large quantities of relativelypure TPFA and its analogues.

It is a further object of the present invention to disclose novelthio-analogues of PFA.

As those skilled in the art will also appreciate, it is also an objectof the present invention to disclose novel methods for converting thegeneral class of phosphonate compounds into thiophosphonates in a simpleand economical manner.

It is yet another object of the present invention to disclose methodsfor inhibiting viral and viral enzyme activities, including those ofHIV, utilizing TPFA.

Lastly, it is a further additional object of the present invention todisclose methods for treating HIV infection in mammalian cells utilizingTPFA or its addition salts as effective antiviral compounds.

SUMMARY OF THE INVENTION

Generally stated, the present invention accomplishes the above-describedobjectives by providing methods for readily converting phosphonates suchas PFA into thiophosphonate analogues such as trimethyl-TPFA in a singlestep reaction which provides unusually high product yields. Moreover,following hydrolysis the TPFA and TPFA analogues produced through themethods of the present invention have unexpectedly high antiviralactivities against HIV while exhibiting unexpectedly low DNA polymeraseinhibiting activity against mammalian enzymes making them particularlywell suited for use as effective anti-HIV agents.

What is more, the processes of the present invention have wideapplicability in converting phosphonates into thiophosphonates for theeconomical production of a wide variety of compounds includinginsecticides incorporating thiophosphonate units. Because the methods ofthe present invention produce desirable thio-analogues of phosphonateswith a synthesis that is short, simple, efficient and which utilizesinexpensive starting materials, the present invention also produces suchcompounds in large quantity at relatively low cost.

More particularly, the methods of the present invention convertphosphonates of the general formula: ##STR3## where R₁, R₂, and R₃ whenpresent as substitutents are each independently hydrogen, hydroxy,methyl, alkyl, aryl, saturated unsaturated. substituted or unsubstitutedorganic compounds, through the following steps.

First, the phosphonate is modified by substituting one or more strongelectron withdrawing groups such as a halogen or doubly-bonded oxygenfor x and y on the alpha carbon adjacent to the phosphorus in thegeneral formula. Those skilled in the art will appreciate that it isunnecessary to modify phosphonate compounds in accordance with theteachings of the present invention where the alpha carbon is alreadybonded to a sufficiently strong electron withdrawing group. It should benoted that, without limiting the scope of the present invention, it isbelieved that the electron withdrawing group or groups must be directlyadjacent to the phosphorus atom of the molecule in order for thereaction to proceed and must be sufficiently strong to drive thefollowing unexpected reactiuon sequence.

The next phase of the method of the present invention involves forming areaction mixture of the modified phosphonate, an effective amount of oneor more forms of Lawesson's reagent (for example, approximately oneequivalent of a dimer of P-methoxyphenylthionophosphine sulfide or othersuitable phosphetane ring containing compounds) and polar, aproticsolvent. An exemplary solvent is acetonitrile or toluene, though thoseskilled in the art will appreciate that any suitable polar aproticsolvent may be utilized. Following the formation of the reaction mixturethe mixture is heated until conversion of the phosphonate into itsthiophosphonate analogue is substantially complete.

Preferably, the heating will take place under an inert, anhydrousatmosphere to prevent interference with the conversion reaction.Exemplary heating temperatures can range from approximately 66° C. to110° C. depending on the solvent utilized and may include refluxconditions. Additionally. heating times may be 1 hour or less, thoughpreferably will be on the order of 2 to 6 hours. In practice, uponheating the reaction mixture, the Lawesson's reagent will be observed togradually disappear and dissolve into the mixture conveniently signalingthat the reaction is progressing to completion.

Once reaction is substantially complete, if desired, the reactionproduct can be separated from the reaction mixture in a variety ofmanners. For example, the solvent can be evaporated and any side product(for example modified Lawesson's reagent, a trimer ofP-methoxyphenylthionophosphine oxide) can be precipitated out of thesolution. Conversely, it is possible to distill the thiophosphonateanalogue from the mixture directly. Using the foregoing methodology anddistilling the product directly from the reaction mixture will produce apure product with yields up to and possibly exceeding 87%. In that thereagents utilized for this essentially one-step reaction are relativelyinexpensive. and the yields of pure product are so high, the economiesof the present invention become readily apparent.

An exemplary phosphonate conversion in accordance with the teachings ofthe present invention utilizes trimethyl phosphonoformate as a startingmaterial to produce trimethyl thiophosphonoformate (O,O-dimethylcarboxymethylphosphonothioate). The trimethyl phosphonoformate is mixedwith approximately one equivalent (±2.5%), of Lawesson's reagent in agenerally two-to-one stoichiometric relationship such that there are twomoles of trimethyl phosphonoformate for every mole of Lawesson'sreagent. The aprotic, polar solvent used is, preferably, eitheracetonitrile or toluene and the mixture is heated under argon for two tosix hours at a preferred temperature of 82° C. for acetonitrile untilthe Lawesson's reagent is observed to dissolve into the mixture.

The trimethyl thiophosphonoformate so produced may be separated from thereaction mixture if desired through precipitation or distillation and,in accordance with the teachings of the present invention, may befurther modified through hydrolysis to produce thiophosphonoformic acid(TPFA) and its addition salts. Preferably, when desired, hydrolysis willtake place under basic conditions such as the utilization ofsodiumhydroxide (NaOH) to directly hydrolyze the trimethyl-TPFA to TPFA.Conversely, though ITMS-H₂ O will not hydrolyze the ethyl-ester of TPFA,it was surprisingly discovered to be effective at hydrolysing the methylester.

The TPFA so produced may then be utilized in accordance with theteachings ofthe present invention as a antiviral inhibitor against HIVvirus and in a method for treating mammals infected with HIV. As will bediscussed in detail below. this unique and unexpected antiviral activityagainst HIV is a product of the high therapeutic index of this compoundwith respect to HIV. More specifically, recent studies made possible bythe method of the present invention show TPFA to be surprisinglyeffective at inhibiting HIV and HIV reverse transcriptase while beingsurprisingly less toxic with respect to inhibition of mammalian DNApolymerase.

Moreover, as will be appreciated by those skilled in the art, theconversion of PFA to TPFA gives the sulfur analogue a lower polaritythus providing enhanced cell-penetration and higher water solubility. Asa result, it is believed that the TPFA antiviral compounds of thepresent invention willbe significantly less toxic than PFA in treatingmammalian cases of HIV infection and inhibit HIV in general.

The above discussed and many other features and attendant advantages ofthe present invention will become apparent to those skilled in the artfrom a consideration of the following detailed description.

DETAILED DESCRIPTION OF THE INVENTION

In a broad aspect, the methods of the present invention are based upontwo surprising discoveries. First, while it is known in the art thatLawesson's reagent (LR) is effective at converting oxygen to sulfur incarbonyl groups such as those found in ketones or in convertingphosphites to thio-phosphites, it was completely unexpected that LRwould prove to be so effective at converting phosphonates tothiophosphonates in accordance with the techniques of the presentinvention. Secondly, the large quantities of pure TPFA so produced madeit possible to determine the relatively high therapeutic index of TPFAwith respect to the inhibition of HIV versus mammalian enzymes; a resultwhich was also completely unexpected in view of the teachings of theprior art. Thus, the methods of the present invention provide new,uniquely effective procedures for rapidly, simply. and inexpensivelyproducing large quantities of essentially pure thiophosphonates. Ofequal or greater significance, the methods of the present invention makeit possible to efficiently produce TPFA and other thio-analogues of PFAin sufficient purity and quantity for use as new, effective antiviralagents against HIV.

Turning first to the general process of converting phosphonates intotheir thiophosphonate analogues and additions salts thereof, a moredetailed understanding of the limitations of the prior art methodologiesfor producing such compounds is in order. The most widely-known methodin the art for reportedly synthesizing thiophosphonates is thatcurrently reported by Hutchinson (D. W. Hutchinson and Masson, Theantiviral potential of compounds containing the thiophosphoryl group,I.R.C.S. Medical Science, 14 (1986) 176-177). Briefly, Hutchinson, etal. report the preparation of alkylthiophosphonate intermediatecompounds utilizing the Michaelis-Becker reaction followed by removal ofthe alkyl groups utilizing iodotrimethylsilane (ITMS). Generally stated,the prior art Hutchison et al. reaction mechanics are reported asfollows: ##STR4##

Additionally, a more limited, alternative prior art synthesis of TPFAwas proposed in a verbal presentation by Dr. William Egan. Though fewdetails are available on this proposed synthesis. it is believed toinclude at least eight to ten steps and may require unique and expensivestarting materials. Thus, as will be appreciated by those skilled in theart, any such synthesis would be a very long, complicated and expensiveprocess having very low overall product yields.

Similarly. the synthesis proposed by Hutchinson et al. is not withoutits own problems. In addition to very low yields, the proposed reactionis incomplete and produces mixtures of unidentified products. Thoughapparently successful at producing some of the intermediate tri.estercompounds, the Hutchinson et al. method apparently is not successful atultimately removing the alkyl groups with ITMS. Elaboration of thesedistinguishing features of the prior art methodology will be providedfollowing a more detailed explanation and understanding of the presentinvention.

As discussed in the foregoing summary section it is known in the artthat Lawesson's reagent (LR) will convert doubly-bonded oxygen to sulfuron carbonyl groups such as those in ketones and will convert phosphitesinto thio-phosphites. There the relative bond energies drive thereaction to completion. However, in the case of phosphonates whereoxygen is doubly-bonded to phosphorus the favorable bond energies arenot present to drive the reaction as LR also includes phosphorus doublebonds. Nevertheless, as disclosed by the present invention. it wassurprisingly discovered that LR would successfully convert phosphonatesinto their thiophosphonate analogues if a sufficiently strongelectron-withdrawing group was located adjacent to the phosphorus in thephosphonate as evidenced by the following non-limiting experimentalreactions.

All reactions were performed in scrupulously dried glassware under N₂.Lawesson's reagent (LR) and Me₃ PFA were used without purification: CH₃CN was distilled from CaH₂ under N₂. All reactions were performed inacetonitrile unless otherwise noted.

REACTION BETWEEN ME₃ PFA and LR

1.66 g (4.11 mmol) LR and 1.38 g (8.22 mmol) Me₃ PFA were suspended in25 ml CH₃ CN and stirred for 2 hours with no apparent reaction. After 12hours, still no reaction had occurred. After heating for 2 hours, the LRdissolved, and NMR indicated that reaction had occurred. Major product,³¹ P NMR: δ=64 ppm. and ³¹ C NMR indicated that the ester group wasintact. Impurities in the ³¹ P NMR spectrum could be removed byevaporating the mixture, and extracting residue between saturated aq.NaHCO₃ and ether, with the product found in the organic layer. In areaction between 1 g ester and 1.3 g LR in refluxing THF for 2 hours.NMR suggested that partial reaction had occurred.

REACTION BETWEEN (tmso)₂ p(O)CO₂ ME AND LR

0.5 g ester and 0.35 g LR were reacted as above for 24 hours; ³¹ P NMRsuggested that reaction had occurred: δ=46 ppm. with impurities. Thesame reaction was performed with 0.5 g ester and 0.7 g LR, and the sameproduct was obtained in a cleaner reaction. Thus, the reaction wasrepeated using 6.5 g ester and 6 g LR, and Proton.coupled ³¹ P NMRshowed that the phosphorus was coupled to a methyl group. (q, ^(J) PH -11 Hz.) CN test was negative. Evaporation of the mixture was done:attempted Kugelrohr distillation failed.

REACTION BETWEEN LR AND OTHER SUBSTRATES

Further experiments conducted as above indicated that LR was inert toEt₃ PFA, Et₃ FPAA, 1PR₄ MDP, 1PR₄ N₂ MDP under the foregoing conditions.However, slightreaction occurred with Et₃ F₂ PAA indicating thatsubstituting strong electron.withdrawing groups such as fluorineadjacent to the phosphorus in the phosphonate compound would drive thereaction to the point that LR would be successful at converting suchmodified compounds into their thiophosphonate analogues. However, itshould be noted that decomposition took place with dibromo and monobromocompounds under the above conditions.

Further demonstrating the utility of the present invention, a variety ofexperiments were conducted utilizing trimethyl phosphonoformate (Me₃PFA) as a starting material for use in accordance with the teachings ofthe present invention. Those skilled in the art will appreciate that Me₃PFA incorporates a strongly electron.withdrawing doubly bonded oxygendirectly adjacent to the phosphorus in the compound which, in accordancewith the present invention, will enable the thio-conversion reaction toproceed utilizing LR. The following non-limiting experiments wereconducted utilizing larger amounts of reagents and under varyingconditions to further illustrate the scope of the present invention.

GENERAL EXPERIMENTAL PROTOCOL

All glassware was scrupu-lously oven, or flame-dried. All reactions wereperformed under dry, pre-purified argon (passed successively throughcolumns of drierite; activated Linde Type 4A molecular sieves; and BASFcatalyst). Lawesson's reagent (LR) was purchased from Aldrich ChemicalCompany (97%) and was used without further purification. Trimethylphosphonoformate (Me₃ PFA) was also purchased from Aldrich ChemicalCompany and was purified by vacuum distillation prior to use (60° C., 15μm).

Solvents were purified using standard methods. Acetonitrile wasdistilled from P₂ O₅, then from CaH₂ ; tetrahydrofuran was distilledfrom benezophenone/sodium ketal, thenfrom lithium aluminum hydride;toluene was distilled from benezophenone/sodium ketal. Hexane and ethylacetate for chromatography were reagent grade and were used directly.Reactions were monitored by thin layer chromatography using silica gel60F-254 (Kieselgel) and detected by an ultraviolet lamp (MineralightUVS-12). Flash column chromatography was performed as described in theliterature (Still, W. C.; Kahn, M.; Mitka, A. J. Org. Chem. 1978, 43,2923.)

NMR spectra were obtained on a Bruker IBM WP-270SY spectrometer operatedat frequencies of 270.02 MHz (¹ H), 109.35 MHz (³¹ P) and 67.92 MHz (¹³C). NMR spectra for isolated compounds were obtained in 5 mm tubes using10% solutions (CDCl₃ for esters. D₂ O for salts). Chemical shifts arereported relative to TMS (¹ H: internal CHCl₃, δ=7.24 ppm; ¹³ C: usinginternal CDCL₃, δ=77.0 ppm); or external 85% H₃ PO₄ (³¹ P). Vacuumdistillations were performed on a vacuum line equipped with an all glassoil diffusion pump; pressures were measured on a MacLeod gauge.

Infrared spectra of esters were obtained on a Perkin.Elmer 281 infraredspectrophotometer. Neat samples were run as thin films between NaClplates. Spectra of salts were taken measured on a FT-IR/32, infraredspectrophotometer

High resolution mass spectra were obtained at the Mass SpectralFacility, University of California, Riverside, Calif.

Elemental analyses were performed by Galbraith Laboratories, Knoxville,Tenn.

EXPERIMENT 1

A 500 mL three.necked round.bottomed flash equipped with refluxcondenser, magnetic stirrer and thermometer was connected to an Arbubbler, flushed with Ar and charged with 375 mL acetonitrile. Me₃ PFA(22.0 g, 130.9 mmol) was added(syringe) and dissolved (magneticstirring). Lawesson's reagent (LR) (dimer ofp-methoxyphenylthionophosphinesulfide)(26.40 g,65.45 mmol)wasadded as apale yellow, chalky powder to the flask (glove bag, Ar). After stirringfor 2 h at room temperature, no reaction or solubilization was evident.The mixture was then refluxed at 82° C. for 6 h, during which time theLR appeared to gradually dissolve, giving a dark-yellow solution. Thereaction mixture was cooled to room temperature causing precipitation ofa creamy white powder. This was removed by suction filtration (9.186 g,fluted filter paper, Eatman Diechmann Grade 515, 2.5 cm) giving a paleyellow solution which was washed and filtered three times with 20 mLdiethyl ether. Excess ether and acetonitrile were removed byevaporation, leaving a pale yellow oil, whose ³¹ P NMR (¹ H) showedsignals at δ=71.9-83.8 ppm and at δ=65 ppm(s); no signal was observed atδ=2 ppm (trimethyl PFA). After 1 day at room temperature, a whitecrystalline solid precipitated from the crude residue. TLC analysis ofthe supernatant (TLC - I), (hexane:ethyl acetate, 6:1) revealed a singlemigrating spot at R_(f) 0.35, in addition to a spot at the sampleorigin. The following summary of analytical TLC results (TLC - II)evaluating different solvents were performed using silica gel 60° F.(Eastman Chromagram), detected by an ultraviolet lamp. In all cases, themixture resolved into two very intense blue mobile spots with nodetection of Me₃ PFA present. In a comparison between Me₃ PFA (R_(f)0.06) and Me₃ TPFA (R_(f) 0.35), the latter was much more intense underultraviolet detection. TLC (hexane:EtOAC, 7:3): Rf 0.07, 0.62, 0.81.(hexane): too weak. (ether): R₅ 0.66, 0.10. (chloroform): no separation.EtOAC: R_(f) 0.10, 0.90. (methylene chloride): R_(f) 0.06, 0.64, 0.90,no strong separation.

The reaction mixture (29 g of total 44 g of crude product) was separatedby flash column chromatography (TLC - I, same solvent system). Likefractions (TLC) were combined and evaporated to yield (³¹ P NMR)virtually pure Me₃ TPFA (O,O - Dimethyl Carboxymethylphosphonothioate)(13.29 g), (contained a trace of impurity with δ=72 ppm). Pure productwas obtained by fractional distillation in vacuo: very pale mobile oil,bp 37.39° C. (10 μm), 10.83 g (87% yield).

More conveniently. the crude supernatant (10 g of 44 g crude product)was directly distilled, giving 4.75 g (87% yield) distillate, pure byTLC, IR, ³¹ P and ¹ H NMR. Bp: 37°-39° C. (10 μm) TLC (hexane:EtOAC,6:1): R_(f) 0.35. ¹ H NMR: δ=3.81 ppm (d, ³ J_(PH) =14 Hz, C(O)OCH₃);3.85 ppm (d, ³ J_(PH) =1 Hz, P(S)OCH₃). ¹³ C NMR: δ=54.3 ppm (qd, ¹J_(CH) =150 Hz, ³ J_(CP) =7 Hz, CH)₃); δ=52.7 ppm (qd, ¹ J_(CP) =149 Hz,³ J_(CP) =5 Hz). ³¹ P NMR: δ=64.8 ppm (septet, ³ ³ J_(PH) =14 Hz). IR:1722 cm⁻¹ (s, v_(CO)), 1035 cm⁻¹ (m, ^(V) POC) no peak at 1290 cm⁻¹(v_(PO), Me₃ PFA). MS: Parent ion m/e 1984, major fragments m/e 125[(CH₃ O)₂ PS⁺ ], m/e 93,79. Parent ion m/e calculated: 183.9959; Found:183.9956. Anal. calculated for C₄ H₉ O₄ PS: C, 26.09; H, 4.93; S, 17.41.Found: C, 26.23; H, 5.00., S, 17.82.

It was shownby TLC that the second fraction collected from flash columnchromatography (R_(f) 0.14) upon evaporation crystallized fromhexane:EtOAC, 4:1. These crystals were very soluble in chloroform andhad the same R_(f) values as the creamy white precipitate that came outof solution and the crystalline solid found on the bottom of the crudeflask. IR and NMR spectra indicate aromatic rings present. A whitediamond-shaped crystal suited for x-ray diffraction crystallography wasmounted on a 0.3 mm glass capillary. Data was collected on afour○Nicolet/Syntex P₂₁ diffractometer employing Cuo radiation. A largetriclinic cell with a volume of 2438 As was revealed by carefullymachine-centering fifteen strong reflections, a procedure which alsoyielded the orientation matrix needed for data collection. The crystaldata is currently being solved. ³¹ P NMR: δ˜7 ppm. ¹ H NMR: δ=72 ppm(m).

Attemqpts to remove byproducts by extractions between saturated aqueousNaHCO₃ and ether were unsuccessful.

EXPERIMENT 2

The same reaction was repeated under identical conditions LR graduallywent into solution and thereaction was refluxed at 82° C. for 6 h.However, upon cooling to room temperature quickly followed byevaporation of acetonitrile, no precipitation was seen. The reactionmixture (57.7 g) was separated by flash column chromatography (TLC I,solvent system) with 10-12 g of crude residue loaded per column. Onecolumn was done with Aldrich silica gel (TLC standard grade, 2-25 μ).Routine TLC - I analysis indicated that in addition to MeTPFAs (R_(f)0.36), another nonpolar intense blue spot was observed (R_(f) 0.46).Like fractions and fractions with mixtures of these two spots werecombined, evaporated and vacuum distilled (bp 38.5° C., 10 μm) yieldingMe₃ TPFA contaminated with impurities (7.84 g, 33% yield). ³¹ P NMR {¹H} of the distillate indicated only pure Me₃ TPFA (δ=64.8 ppm) howeverTLC analysis still showed two spots. A further TLC - I analysis of theimpurity (R_(f) 0.56) was made by varying the amount of sample spottedon the plate. The plate was observed under an ultraviolet lamp and thentreated with phosphomolybdic acid (spray, 7% solution in ethanol).Examination of the TLC plate revealed that this compound consisted of alight blue spot (R_(f) 0.56) in addition to a dark violet-blue spot(R_(f) 0.39), the latter of which has a similar R_(f) value to Me₃ TPFA(R_(f) 0.35) which appeared to be a faint blue color. ³¹ P NMR {¹ H}showed a signal at δ=99.99 ppm. This impurity was carefully removed byflash column chromatography (TLC - I, solvent system). A long columnvacuum distillation yielded pure Me₃ TPFA (6.50 g, 27% yield, bp 39.5°C., 10 μm).

EXPERIMENT 3

Several reactions wererun changing the reactants addition order. In a100 mL round-bottomed flask, LR (5.3 g, 13.09 mmol) was added under Arand charged with 75 mL acetonitrile (glove bag, Ar). No immediatereaction or solubilization was evidentwith stirring. Me₃ PFA (4.4 g, 2618 mmol) was added (syringe) and the mixture was refluxed at 82 ° C. for6 h at which time LR appeared to gradually dissolve giving a dark yellowsolution. When the reaction mixture was cooled to room temperature, noprecipitation was seen. Only after evaporation of acetonitrile, acrystalline solid precipitated from the crude residue which wasidentical to thesolid described in the experiment previously (TLC - I).Flash column chromatography (TLC - I. solvent system) and vacuumdistillation yielded pure product, Me₃ TPFA (2.78 g, 57% yield).

EXPERIMENT 4

In a similar reaction, LR (5.3 g, 13.09 mmol) was added under Ar andcharged with 75 mL THF (glove bag, Ar). Again, no immediate reaction orsolubilization was evident with stirring. Me₃ PFA (4.4 g, 26.18 mmol)was added (syringe) and the mixture was refluxed at 66° C. LR went intosolution within 2 h. Refluxing was continued for 4 h more. NMR indicatedno starting material left. Again, the crystalline solid precipitatedafter 1 day standing at room temperature. Pure product was obtainedafter flash column chromatography (TLC - I, solvent system) anddistillation in vacuo, Me₃ TPFA (2.20 g, 48% yield).

EXPERIMENT 5

In a similar reaction, excess LR (22.62 g, 55.03 mmol) was added underAr to a 500 mL round-bottomed flask and charged with 375 mL acetonitrile(glove bag, Ar). Again, no immediate reaction or solubilization wasevident with stirring Me₃ PFA (17.56 g, 104.4 mmol) was added (syringe)and the mixture refluxed at 82° C. for 6 h at which time the LR appearedto gradually dissolve giving a yellow solution. The reaction mixture wascooled to room temperature and excess acetonitrile was removed byevaporation leaving a pale yellow oil with slight precipitation of acreamy white powder. After one day standing at room temperature, a largeamount precipitated (39.00 g) containing a small amount of yellow oil.The oil was found to be very soluble in hot hexane leaving the solidresidue behind. To the crude residue, 200 mL hexane was added and heatedwith steam until reflux and gravity filtered while hot (steamfiltration, Whatman filter paper Grade #1). Excess hexane was removed byevaporation giving a pale yellow oil (13.48 g). TLC analysis oftheyellow oil (hexane:ETOAC, 4:1) revealed intense blue spots (R_(f) 0.46,0.26, 0.04) under an ultraviolet lamp Analysis of the precipitate showedtwo intense blue spots (R_(f) 0.2, 0.04) and two spots which werelighter blue (R_(f) 0.56, 0.44). Direct distillation of the crude yellowoil gave 11.63 g (61% yield) distillate (bp 38°-39° C., 10 μm). TLC and³¹ P NMR revealed this to be Me₃ TPFA (δ=64.8 ppm) with a little Me₃ PFAstarting material (6%). The crude residue left in the pot was a thicklemon yellow gum (bp>100° C.) having the same properties as the LR sideproduct discussed earlier (TLC).

EXPERIMENT 6

Three reactions were performed simultaneously using toluene as thesolvent varying the relative amounts of LR used. A 100 mL, and two 250mL round.bottomed flasks were flushed with Ar and charged with 5 mLtoluene. To each flask, Me₃ PFA (4.4 g. 26.18 mmol) was added (syringe)and dissolved (magnetic stirring). Consecutively, LR (5.6 g, 3.72 mmol)was added to the 100 mL flask (I), LR (11.1 g, 27.4 mmol) was added toone 250 mL flask (II), and LR (16.7 g, 41.2 mmol) was added to the other250 mL flask (III), (glove bag, Ar). Again. no immediate reaction orsolubilization was evident with stirring. The mixtures were refluxed at100° C. and LR gradually dissolved giving yellow solutions.

All reactions were stopped after 1 h. I was a pale yellow solution withno precipitate present even after cooling to room temperature (similarto the THF reaction). II was a clarified lemon-yellow solution whichprecipitated a pale yellow crystalline residue at room temperature. IIIwas a darker lemon-yellow solution with a large amount of lemon-yellowprecipitate. A summary of TLC results (hexane:ETOAC, 7:3) monitoring thereactions were detected by an ultraviolet lamp. I showed very little ofboth Me₃ PFA (R_(f) 0.08) and MeTPFAs (R_(f) 0.5) but an intense bluespot similar to the LR crystalline side product previously described(R_(f) 0.32). II and III showed similar results, however, more Me₃ PFAwas present for these two, I, II and III had similar ³¹ P NMR (¹ H)showing signals for MeTPFAs δ=65 ppm(s) and signals at δ=4, 71.8-75.1,and 92.3 ppm.

Refluxing was continued for 1 h and after standing at room temperatureovernight I was a pale yellow solution, II was a clarified lemon-yellowsolution with a pale yellow crystalline residue and III was a darkerclarified lemon-yellow solution with a large amount of crystallineprecipitate. TLC analysis (hexane:ETOAC, 7:3) showed an intense bluespot for all three (R_(f) 0.34) similar to the LR crystalline sideproduct, a light blue spot (R_(f) 0 56) similar to MesTPFA. in additionto two lightblue nonpolar spots (R_(f) 0.62, 0.96). Both II and IIIstill showed some Me₃ PFA present (R_(f) 0.5). ³¹ P NMR (¹ H) of I, IIand III revealed the peaks at δ>71 ppm. Analysis of I showed the singletat δ=65 ppm now was a multiplet (δ=65.4-65.6 ppm). Similarly, analysisof II showed a doublet (δ=65.3-65.4 ppm) III showed no change (δ=65.5ppm).

From the foregoing experiments, it will be apparent to those skilled inthe art that the methods of the present invention are particularlyeffective at producing the thio analogue of Me₃ PFA, namely trimethylthiophosphonoformate (O,O-Dimethyl Carboxymethylphosphonothioate).Moreover, the methods of the present invention produce this compoundwith exceedingly high yields in a very simply, essentially one.stepreaction utilizing inexpensive starting materials. As detailed above,the trimethyl thiophosphonoformate can be readily separated from thereaction mixture through distillation or precipitation orchromatographic methodologies.

It is also contemplated as being within the scope of the presentinvention to utilize the additional step of hydrolyzing the trimethylthiophosphonoformate to produce thiophosphonoformic acid and/or itsaddition sales. Preferably, hydrolyzation will take place under basicconditions as illustrated by the following non-limiting examplesdetailing the production of TPFA and its sodium addition salt. However,those skilled in the art will appreciate that other hydrolyzationmethods including the correct usage of ITMS are contemplated as beingwithin the scope of the present invention

EXPERIMENT 7

2.75 ml of 10 N sodium hydroxide solution were added to 1.0 g (5.43mmol) of Me₃ TPFA with vigorous stirring at room temperature. After 3.5minutes, the mixture became hot and the methanol produced evaporated.Stirring was continued for ca. 15 min. and the mixture cooled in an icebath. The pH was adjusted to 10.5 with 1N HCl. The solvent wasevaporated by dry.ice freeze pumping. Distilled water (2 mL) and excessmethanol were added. The precipitate formed was centrifuged andover.dried in vacuo, neutralized to pH 6 with 1N HCl then readjusted topH 10.5 with 1N NaOH. The solvent was again evaporated by dry-ice freezepumping. Then 1.5 ml water and excess methanol were added. Theprecipitate formed was centrifuged and oven. dried in vacuo. The processwas repeated, 207.5 mg of pure desired salt were obtained (18.4% yield)³¹ P NMR: δ=37.7 ppm (s). ¹ H NMR: δ=4.63 ppm (H₂ O) in D₂ O :¹³ C NMR:δ=183.2 ppm (d, ¹ J_(CP) =181 Hz, CO). IR. Anal. Calculated for: C 5.77;H, 0.00; S, 15.41. Found: C, 5.83; H, 0.12; S, 14.91.

Those skilled in the art will appreciate that while the cleavage ofester compounds through basic hydrolysis is known in the art, whether ornot a particular di.functional ester will be cleaved under suchconditions cannot be predetermined. Thus, as further evidence of thescope of the present invention, it is possible to substitute thedi.ethyl ester of TPFA for Me₃ TPFA in the above experiment to produceTPFA. As expected, when utilizing Et₃ TPFA as a starting material, someof the foregoing reaction conditions for base hydrolysis must bechanged. For example, the reaction time must be increased toapproximately thirty minutes and it is also anticipated that theproduction of side products may be increased.

EXPERIMENT 8

An improved yield of pure Na₃ TPFA was obtained utilizing the followingprotocol. As before 2.75 ml of 10N sodium hydroxide solution were addedto 1.0 g (5.43 mmol) of Me₃ TPFA with vigorous stirring at roomtemperature. After 3.5 minutes, the mixture become hot and cloudy, andmost of the methanol produced evaporated. Stirring was continued forapproximately 10 min., and the mixture was cooled in an ice bath.Distilled water (3 mL) and 30 mL methanol were added. The precipitateformed was centrifuged and oven-dried in vacuo (<1 mm Hg, 50° C.) 10min., neutralized to pH 4.5 to remove CO₂ (from Na₂ CO₃ formed duringthe reaction), with 3N HCl (approximately 4-5 mL) then readjusted to pH10.5 with 3N NaOH (approximately 0.5 mL). The solvent was evaporatedbylyophilization. Water (2.5 mL) and methanol (30 mL) were then added. Theprecipitate formed was centrifuged as before and oven-dried in vacuo (<1mm Hg 55° C. for 6 h). The process was repeated. 231.5 mg of pure Na₃TPFA (white powder) was obtained (20.5% yield). ³¹ P NMR: δ=37.7 ppm(s). ¹ H NMR: no resonances other than HDO. ¹³ C NMR: δ=183.2 ppm (d, ¹J_(CP) =181 Hz. CO). IR: 1680 cm⁻¹ (m), 1095 cm⁻¹ (shoulder), 1580 cm⁻¹(s), 1375 cm⁻¹ (s), 1140 cm⁻¹ (s), 1030 cm⁻¹ (s). UV: .sup.δ 254nm=1.05×10³, .sup.ε 233=2.44× 10³, .sup.ε 205=6.0×10³. Analyticalcalculated for Na₃ TPFA: C, 5.77; H, 0.00; S, 15.41. Found: C, 5.83; H,0.12; S, 14.91.

It should be noted that by using Et₃ TPFA as starting material, the sameproduct can be obtained, but in low yield and accompanied by impurities.Additionally, when using Et₃ TPFA as a starting material the reactiontime needed to be increased to approximately 30 minutes. resulting inincreased formation of side product.

Nonetheless, as will be appreciated by those skilled in the art, thereagents utilized in the foregoing basic hydrolysis experiments are veryinexpensive relative to compounds such as ITMS, further contributing tothe superior economics of the methods of the present invention.Moreover, as detailed in the following examples, the prior artmethodologies utilizing expensive reagents such as ITMS are notsuccessful at producing thiophosphonates such as TPFA.

In the following examples, a variety of phosphonate starting materialswere synthesized utilizing either the method of the present invention orthat of Hutchinson et al. where possible. These compounds were thensubjected to ITMS hydrolysis as disclosed in the prior art to illustratethe difficulties of this prior art methodology and to furtherdistinguish the novel methods of the present invention.

PREPARATION OF O,O-DIETHYL HYDROGEN PHOSPHOROTHIOIITE [EtO)₂ P(S)H](Michaelis-Becker Reaction)

A mixture of diethyl dithiophosphate and triphenylphosphine was stirredvigorously at 65° C. for 7 h. After fractional distillation in vacuo,the product was obtained in 59% yield. Bp: 61°-62° C. (4 mm). ³¹ p NMR:δ=69.3 ppm (dp, ¹ J_(PH) - 647 Hz, ² J_(PH) - 11 Hz). ¹ H NMR: δ=1.16ppm (t, ³ J_(HH) - 7 Hz, CH₃ CH₂); δ=3.98 ppm, (m, ³ J_(HH) - 7 Hz, CH₃CH₂); δ=7.57 ppm (d, ¹ J_(HP) - 647 Hz, P(S)H). ¹³ C NMR: δ=15.69 ppm(qd, ¹ J_(CH) - 204 Hz, ³ J_(CP) - 12 Hz, CH₃ [PO]); δ=61.78 ppm (t, ¹J_(CH) -238 Hz).

PREPARATION OF Et₃ TPFA cl (Michaelis-Becker Reaction)

Finely divided sodium was suspended in dry benzene. The suspension wasadded to the solution of diethyl thiophosphite also dissolved inbenzene. The mixture was heated to 50° C. for 2 h, then cooled to 5° C.with ice bath. Ethyl chloroformate dissolved in benzene was addeddrop.wise to the above mixture at room temperature. The mixture washeated to 50° C. for 3 h, then cooled to room temperature, andcentrifuged. The clear resulting solution was washed with water, driedwith MgSO., evaporated, and separated by fractional distillation invacuo. The yield was 43%. Bp: 95°-96° C. (1.22 mm). IR. ³¹ P NMR: δ=61.4ppm, (P, ³ J_(PH) - 10 Hz). ¹ H NMR: δ=1.25 ppm (m, ³ J_(HH) -7 Hz,CH₃), 9H; δ=4.17 ppm (m, ³ J_(HH) - 7 Hz, CH₂), 6H. ¹³ C NMR: δ=13.7 ppm(q, ¹ J_(CH) - 128 Hz, CH₃ [CO]); δ=15.8 (qd, ¹ J_(CH) - 128 Hz, ³J_(CP) - 7 Hz, CH₃ [PS]); δ=62.1 ppm (td, ¹ J_(CH) - 145 Hz, ³ J_(CP) -4 Hz, CH₂ [CO]); δ=64.2 ppm (td, ¹ J_(CH) - 149 Hz, ³ J_(CP) - 7 Hz, CH₂[PS]); δ=167.3 ppm (d, ¹ J_(CP) - 225 Hz, CO). Anal. Calculated for: C,37.16; H, 6.68; S, 14.17. Found: C, 36.9; H, 6,62; S, 14.06

PREPARATION OF METHYL (O,O-DIMETHYL) THIOPHOSPHONOFORMATE (ME₃ TPFA)(Present Invention Methodology)

LR and Me₃ PFA were suspended in CH₃ CN or THF and heated ro 78° C. for6 h and reaction was followed to TLC. The reaction mixture was thenevaporated in vacuo; the components were separated by silica gel columnusing a mixture of hexane and ethyl acetate (6:1) as eluting solvents.The progress of the separation followed by TLC. The fractions werecollected, combined, evaporated, and distilledin vacuo. The yield was87%. Bp: 37°-38° C. (10 μm). IR. ³¹ P NMR: δ=64.8 ppm (p, ³ J_(PH) =14Hz). ¹ H NMR: δ=3.81 ppm (d, ⁴ J_(HP) =1, Hz⁶, CH₃ [CO]); δ=3.83 ppm (d,³ JHP =14 Hz, CH₃ [PO]). ¹³ C NMR: δ=54.3 ppm (qd, ¹ J_(CH) =150 Hz, ³J_(CP) =7 Hz, CH₃ [PS]); δ =52.7 ppm (qd, ¹ J_(CH) =149 Hz, ³ J_(CP) =5Hz, CH₃ [CO]); δ=167.2 ppm (d, ¹ J_(CP) =226 Hz, CO). Mass. M/e: 184(FW: 184.15). Anal Calculated for: C, 26.09; H, 4.93; S, 17.41. Found:C, 26.23., H, 5.00., S, 17.82.

PREPARATION OF BENZYL (O,O-DIETHYL) THIOPHOSPHONOFORMATE(Michaelis-Becker Reaction) ##STR5##

A finely divided sodium was suspended in dry benzene. The suspension wasthen added to a solution of diethyl thiophosphite in benzene. Themixture was heated to 50° C. for 1.5 h, then cooled to 5° C. in icebath. Benzyl chloroformate dissolved in benzene was added drop.wise tothe above mixture at room temperature. The resulting mixture was heatedto 50° C. for 2.5 h, washed with H₂ O, dried with MgSO₄, evaporated, anddistilled in high vacuo. The yield was 48%.¹ Bp: 130°-132°0 C. (4 μm).IR. ³¹ P NMR: δ=61.2 ppm (p, ³ J_(PH) =10 Hz). ¹ H NMR: δ=1.196 ppm (t,^(3J) HH =7 Hz), CH₃ [PS]; δ=4.132 ppm (m, ³ J_(HH) =7 Hz), CH₂ [PS];δ=5.025 ppm (s), CH₂ [CO]; δ=7.230 ppm, C₆ H₅ [CO]. ¹³ C NMR: δ=16.0 ppm(qd, ¹ J_(CH) =128 Hz, ³ J_(CP) =7 Hz, CH₃ [PS]; δ=64.6 ppm (td, ¹J_(CH) =149 Hz, ³ J_(CP) =7 Hz, CH₂ [PS]); δ=69.5 ppm (t, ¹ J_(CH) =148Hz, CH₂ [CO]); δ=128.4 ppm (d, ¹ J_(CH) =159 Hz, C₆ H₅ [CO]); δ=167.3ppm (d, ¹ J_(CP) =224 Hz, CO).

The compounds so produced were then subjected to ITMS hydrolysis asdisclosed by Hutchinson et al. with the following results:

USING ME₃ TPFA AS STARTING MATERIAL

On a small scale, the prior art method was successful at obtaining thedesired product though it was expensive and time consuming. Utilizingclassical aqueous TMS ester quenching conditions xcess ITMS was added toMe₃ TPFA at room temperature with stirring under N₂. The mixture washeated to 110° C. (oil bath) for 5 h and the reaction progress followedby ³¹ P NMR Excess ITMS was removed under high vacuum and ³¹ P and ¹ HNMR showed that the reaction was complete. ##STR6##

The residue was cooled in an ice bath. Cold water was added and themixture stirred for 10 min. It was then titrated to pH 10.50 with NaOHsolution. Excess solvent was evaporated by lyophilization. Methanol wasadded, and the mixture centrifuged. The product was oven.dried undervacuum producing a low yield of less than 10%.

When the hydrolyzed crude product was carefully analyzed, adecarboxilation reaction was also discovered: ##STR7##

However, when increased amounts of Me₃ TPFA (2g) were used in thereaction, thereby necessitatinglonger reaction times (e.g., refluxingfor 15 h) the reaction was not complete and the decomposition productquickly increased.

USING Et₃ TPFA AS STARTING MATERIAL

Dealkylation of Et₃ TPFA by treatment with ITMS was more difficult thanthat of MeTPFA.s A mixture of Et₃ TPFA and excess ITMS was refluxed at120° C. (oil bath) and the reaction progress was followed by ³¹ P NMR.

After 3 h, an intermediate was obtained: ##STR8##

After 7 h, the didealkylation produced was obtained: ##STR9##

Accordingly, the reaction mechanism was presumed to be as follows:##STR10##

It should be noted that it was very difficult to remove the C₂ H₅ fromthe carbonyl group because continued reflux with ITMS caused the sideproducts to increase quickly. The reaction was repeated in the Presenceof an alkyl iodide in an attempt to use a Pishchimuka thiono-thiolorearrangement to obtain the thiolo analogue prior todealkylation.silylation with ITMS. Unfortunately, even after refluxingEt₃ TPFA with 1.iodobutane in nitromethane for 44 h, only about 35% ofthiolo analogue could be obtained (³¹ P,δ=26.9 ppm). Continued refluxingcaused decomposition.

Further demonstrating the distinguishing features of the presentinvention over the teachings of the prior art, the followinghydrolyzation methodology was developed in accordance with the teachingsof the present invention in order to hydrolyze Me₃ TPFA using ITMS whileavoiding the problems of decarboxylation and decomposition associatedwith the prior art methodologies.

USING Me₃ TPFA AS STARTING MATERIAL

2.5 mL (ca. 16 mmol) ITMS was added to 500 mg (2.72 mmol) of Me₃ TPFA atroom temperature with stirring under N₂. The mixture was heated to115°-120° C. for 7 h and the reaction progress followed by ³¹ P NMR.Excess ITMS was then removed under high vacuum (0.001 mm Hg) at roomtemperature. The residue was cooled in an ice bath and was then added to318 mg (3 mmol) of Na₂ CO₃ in 2 mL water and dissolved with stirring.The mixture was adjusted to pH 10.5 using 3N NaOH solution(approximately 0.5 mL) On addition of 35 mL of methanol, a precipitateformed which was centrifuged 10 min. and dried in a vacuum oven (<1 mmHg at 50° C.), neutralized to pH 4.5 with 3N HCI (approximately 4 mL),then readjusted to pH 10.5 with 3N NaOH (ca. 0.5 mL). Reprecipitationwith 35 mL of methanol, centrifugation as above, and oven drying invacuo (<1 mm Hg, 50° C.) gave a white precipitate. This was redissolvedin 3 mL of water, and the same procedure repeated three times. Afterdrying (<1 mm Hg, 55° C., 6h), 243.9 mg (43% yield) NasTPFA, (whitepowder) was obtained. ³¹ P NMR: δ=37.7 ppm (s). ¹ H NMR: no resonancesexcept HDO. ¹³ C NMR: δ=183.3 ppm (d, ¹ J_(CP) =181 Hz, CO).

Accordingly, from the foregoing it will be readily apparent to thoseskilled in the art that the prior art method of Hutchinson et al., whilemarginally successful at producing some intermediate compounds, does notproduced TPFA or its addition salts. Apparently, the conversion of thephosphonate to the thiophosphonate compound interferes with thereactivity of ITMS. As a result, mixtures are produced by the prior artrather than pure compounds. As those skilled in the art will alsoappreciate, the significant questions raised as to the results of thereported synthesis by Hutchinson et al. also raise doubts as to thereproducability or veracity of any antiviral data that may have beenpublished with respect to PFA or TPFA.

In contrast, the method of the present invention is highly successful atinexpensivelyproducing large yieldsof pure TPFA thereby enabling itsantiviral properties to be properly ascertained. More particularly, asshown in the following, non-limiting examples, in accordance with theteachings of the present invention, TPFA is unexpectedly effectiveagainst both HIV and HIV reverse transcriptase.

Inhibition of a variety of viral enzymes was measured in order todetermine the IDss or Inhibitory Dosage 50 of TPFA versus itsphosphonate analogue PFA utilizing the following protocol.

PREPARATION OF DNA POLYMERASES

Viral DNA polymerases (HSV-1, HSV-2, EBV) were purified by previouslypublished methods (Derce, D. K. F. Bastow, and Y. C. Cheng (1982) J.Boil. Chem. 257:10251-10260; Ostrander, M., and Y. C. Cheng (1980)Biochim. Biophys. Acta 609:232-245; Tan, R. S., A. Datta. and Y. C.Cheng (1982) J. Virol. 44:893-899). These procedures generally includedsequential chromatography on DEAE-cellulose. HIV reverse transcriptasewas purified by antibody affinity column chromatography as descrbed(Starnes. M. C. and Y. C. Cheng (1989) J. Biol. Chem. 264:7073-7077).The purified enzymes were dialyzed against and stored in 50 mM Tris-HCl(pH 7.5) containing 1 mM each of DTT, EDTA and PMSF plus 30% glycerol.Mammalian DNA polymerases alpha, beta, gamma, and delta were partiallypurified from K562 celles (chronic myelogenous leukemia tissue cultureline). Briefly, washed cell pellets were extracted and passed throghDEAE-cellulose in the presence of 300 mM KPO₄ (pH 7.5) as previouslydescribed (Starnes, M. C., and Y. C. Cheng (1987) J. Biol. Chem282:988-981) to remove DNA. The column flow-through fractions weredialyzed against buffer which contained 50 mM Tris-HCl (pH 7.5), andfractionated on a single-stranded DNA-cellulose column with a 0-1M KClgradient. Mammalian polymerases (peak fractions) were completelyseparated from each other and exhibited the typical inhibitor profileand associated enzyme activities for each enzyme (effect of aphidicolin,dideoxynucleotides, butyiphenyl-dGTP, and ionic strength, presence ofprimase and reverse transciptase activity).

HIV-1 REVERSE TRANSCRIPTASE ASSAYS

Standard assays were run at 37° C. and contained: 50 mM Tris, pH b 8.0,0.5 mM DTT, 8 mM MgCl₂, 100 μg/mL BSA, 150 μg/mL gapped calf thymus DNA,100 μM each dATP, dCTP, dGTP, 10 ∥M [³ H]-dTTP, and 1-5 ∥L enzyme in afinal volume of 50 ∥L. Modified assays for pH dependence inhibitionstudies with PFA (1) and α--oxo phosphonates (4 and 5) contained 50 mMHepes, pH 8.2-6.5, 8 mM MgCl₂, 100 mM KCl, 100 μg/ml BSA, 0.5 A₂₆₀units/ml of poly(rA).(dT)₁₀, 100 μM [³ H]dTTP, and 1.5 ∥L enzyme in afinal volume of 50 μL. Samples were processed as described above.

The results of these assays were tabulated as follows:

                  TABLE I                                                         ______________________________________                                        Viral Polymerase Inhibition                                                                 ID.sub.50 (μM)*                                              Virus           TPFA    PFA                                                   ______________________________________                                        HIV-1            1      0.7                                                   HSV-1             11.8  0.7                                                   HSV-2             11.3  0.7                                                   EBV             70      1                                                     HV-6            70      1                                                     ______________________________________                                         *All assays with "activated" DNA.                                        

As those skilled in the art will appreciate, from the foregoing the ID₅₀with respect to HIV for TPFA is essentially identical to that for PFA.This is in startling contrast to the effectiveness of TPFA relative toPFA with respect to the other virus enzymes tested. As shown in Table ITPFA is fifteen to twenty times less effective than PFA with respect toHerpes Simplex virus Type I and II and more than seventy times lesseffective against Epstein-Barr virus and Herpes Virus 6. Yet TPFA isequally effective against HIV. Thus, as will be appreciated by thoseskilled in the art, while the antiviral inhibitory activity of TPFA iscompletely unpredictable it is also surprisingly effective against HIV.

Moreover, the test results shown in Table I indicate that the previouslyreported inhibitory effects of TPFA and PFA are incorrect. Morespecifically, Hutchinson et al. reported relative ID₅₀ values for PFAand TPFA with respect to HSV-1 of, respectively, 12 and 9. In that thepure compounds of the present invention tested in Table I show adifference in inhibition activity with respect to HSV-1 on the order ofa factor of 12 between TPFA and PFA, it would appear that Hutchinson etal. who report on essentially identical activity between the twocompounds were most likely measuring mixtures rather than purecompounds. Thus, because of the prior art difficulties in producingTPFA, it is clear that the previously reported antiviral activities ofTPFA are incorrect.

In accordance with the teaching of the present invention similar enzymeinhibition assays were also conducted with respect to mammalian enzymes,more particularly, human DNA polymerase. The results of these tests aretabulated as follows:

                  TABLE II                                                        ______________________________________                                        Human DNA Polymerase Inhibition                                                      % Control at 100 μM ± S.D. (ID.sub.50, μM).sup.a              Pol      TPFA          PFA                                                    ______________________________________                                        α  72 ± 3 (>100)                                                                            16 ± 2 (31)                                         β   91 ± 4 (>100)                                                                            89 ± 8 (>100)                                       γ  75 ± 3 (>100)                                                                            55 ± 5 (>100)                                       δ  69 ± 6 (>100)                                                                            36 ± 3 (71)                                         ______________________________________                                         .sup.a All assays with "activated" DNA.                                  

As shown in Table II the estimated ID₅₀ of TPFA with respect to thesemammalian enzymes is significantly higher than that for PFA, especiallywith respect to human DNA polymerase-Alpha, the most important DNApolymerase in this comparison. Even more significantly, TPFA is greaterthan 100 fold less active against the human enzyme than it is againstthe HIB enzyme tested in Table I. Accordingly, the therapeutic index forTPFA is relatively high. In fact, as shown in Tables i and II thetherapeutic index for TPFA with respect to HIV enzyme and human enzymeis such that TPFA is, at a minimum, three times less toxic to the humanenzyme than PFA yet is equally effective against the viral enzyme.

In addition to testing the activity of TPFA against HIV enzyme, theactivity against the virus itself was also determined in accordance withthe following protocol. The experimental protocol involved incubatino ofH9 lymphocytes (3.5×10⁶ cells/ml) in the presence or absence of HIV-1(HTLV-IIIB) for one hour at 37° C. Cells were washed thoroughly toremove unabsorbed virions and re-suspended at 4×10⁵ cells/ml in culturemedium. Aliquots (1 ml) were placed in wells of 24 well culture platescontaining any equal volume of test compound (diluted in culturemedium). After incubation for three days at 37° C., cell density ofuninfected cultures was determined to assess toxicity of the testcompound. A p24 antigen capture was used to determine the level of HIVinfection in HIV treated cultures. The ability of test compounds toinhibit HIV replication was measured at different concentrationsrelative to infected, untreated cultures. Test compounds were consideredto be active if p24 levels were <70% of infected, untreated cultures.Cytotoxicity in uninfect4ed H9 cells was not detected.

The results from this experimental data were tabulated in the followingtable:

                  TABLE III                                                       ______________________________________                                        HIV Inhibition In Cell Culture.sup.a                                                           Cell                                                         Drug  Drug Conc. Survival        p24 Ave                                                                              Inhibition                            Type  (μg/mL) (%)      Toxicity                                                                             (%)    Score.sup.b                           ______________________________________                                        PFA   120        102      non-T  -2     ***                                         30          92      non-T  10     ***                                         7.5         96      non-T  35     **                                          1.9         98      non-T  88                                           TPFA  200         84      non-T  -3     ***                                         50          93      non-T   0     ***                                         10         104      non-T  35     **                                          2          110      non-T  90                                                 0.5        113      non-T  117                                                0.1        113      non-T  139                                          ______________________________________                                         .sup.a p24 antigen capture assay.                                             .sup.b ***, strong; **, moderate; *, weak.                                    REMARKS:                                                                      Control untreated H9 cell count  1.07 million                                 Control infected H9 p24 = 357 μg/mL                                        Drug concentration μg/mL                                                   Toxic:                                                                        "nonT" >70% cell survival                                                     "T" <70% cell survival                                                        Score:                                                                        *50-69% control p24                                                           **25-49% control p24                                                          ***<25% control p24                                                      

As those skilled in the art will appreciate, in Table III the lower thevalue of p24 the more active the compound is at inhibiting HIV. As shownin Table III, TPFA is as effective at inhibiting HIV at 50 μg/mL as PFAis at 120 μg/mL. Coupling these results with the previously discussedtherapeutic index of TFPA, it becomes abundantly clear that TPFA isunexpectedly superior to its phosphonate analogue PFA with respect toinhibition of HIV. What is more, as those skilled in the art will alsoappreciate, there is good reason to believe that TPFA will besignificantly less toxic in treating mammals as well as more effective.For example, TPFA exhibits a lower molecular polarity enhancing itswater colubility and cell penetration properties. Thus, unlike PFA whichmay crytalize in the kidneys causing a toxic reaction, the highersolubility compound TPFA is less likely to crystalize and cause suchside effects. Additionally, because the thiophosphonate compound is lesslike a phosphate compound it should exhibit a reduced tendency todeposit in bone.

Moreover, as those skilled in the art will also appreciate, TPFA and/orits addition salts can be administered to mammals including humans in aneffective amount as determined by clinical trials. The compound may beadministered orally, parenterally, topically or by other standardadministration routes. Additionally, the compound can include apharmaceutically acceptable carrier such as the normally acceptableadditives, excipients, and the like and may also be combined with otherbioreactive compounds such as AZT, DDC, DDI and antibiotics.

An additional advantage of TPFA over PFA is that the ultravioletspectrum of TPFA is about ten times more intense than that of PFA and iseasier to detect. The presence of sulfur in TPFA tends to shift theultraviolet absorption towards the red-end of the spectrum making itmore convenient to measure. As a result, the TPFA produced in accordancewith the teachings of the present invention has additional analyticalbenefits for chemical research.

Having thus described exemplary embodiments of the present invention, itshould be noted by those skilled in the art that the within disclosuresare exemplary only and that various other alternatives, adaptations andmodifications may be made within the scope of the present invention. Forexample, solvents other than acetonitrile or toluene may be utilized aswell as other inert gases in place of the argon disclosed and claimed.Additionally, other phosphonate starting materials may be utilized thanthose disclosed in the foregoing non-limiting examples. Accordingly, thepresent invention is not limited to the specific embodiments asillustrated herein, but is only limited by the following claims.

What is claimed is:
 1. A method for the production of thiophophonoformicacid, said method comprising the steps of:forming a reaction mixture oftrimethyl phosphonoformate, an effective amount of Lawesson's reagentand polar, aprotic solvent; heating said reaction mixture untilconversion of trimethyl phosphonoformate to trimethylthiophosphonoformate is substantially complete; and hydrolysing saidtrimethyl thiophosphonoformate to form thiophosphonoformic acid.
 2. Themethod of claim 1 further comprising the additional step of:separatingsaid trimethyl thiophoosphonoformic acid from said reaction mixtureprior to hydrolysis.
 3. The method of claim 2 wherein said trimethylthiophosphonoformic acid is separated from said reation mixture throughdistillation.
 4. The method of claim 1 wherein said polar, aproticsolvent is acetonitrile.
 5. The method of claim 1 wherein said reactionmixture is heated under an inert, anhydrous atmosphere.
 6. The method ofclaim 1 wherein said trimethyl thiophosphonoformate is hydrolysed underbasic conditions.